莲与美洲黄莲花色分化的分子机理

发布时间：2018-02-06 07:43

本文关键词： 莲美洲黄莲花色花青素 MYB GST　出处：《中国科学院研究生院(武汉植物园)》2016年博士论文　论文类型：学位论文

【摘要】：莲是一种集观赏、药用和食用于一体的水生植物。莲属包含两个物种,即莲(Nelumbo nucifera Gaertn.)和美洲黄莲(N. lutea Pers.)。两物种虽然间断分布于东亚-北美和澳大利亚北部,但是不存在生殖隔离,杂交可育。除此之外,两物种在形态上差异不明显,最显著的差异是花色。莲具有红色、粉色、白色和红白相间四种花色,独缺黄色,而美洲黄莲只有黄色。有关莲属花色的分子机理非常少,目前仅有红、白花色差异的研究,没有两物种间花色差异的研究。究竟是什么导致了亲缘关系相近的莲与美洲黄莲在花色上差异如此显著?为了解开这个谜底,本研究以黑龙江的野生莲(红色花)和美国佛罗里达州的野生美洲黄莲(黄色花)为材料进行研究。首先,本研究采用分光光度计法测量了红、黄两种花瓣中花青素的含量。然后,通过基因克隆、本地blast和系统发育树的构建,找到在莲属中参与花青素合成及转运的结构基因和调节基因。通过比较这些基因在两物种中的核苷酸序列及转录水平,找到可能参与花色差异的候选基因,并结合酵母双杂和转基因技术进行验证。取得的主要结果如下：(1)野生莲的红色花瓣中花青素的含量较高,而美洲黄莲的黄色花瓣中几乎不含花青素。这表明美洲黄莲花瓣中花青素合成途径可能发生中断。波长扫描显示在350nm处,两种花色的花瓣提取物都有一个高的吸收峰,表明黄酮和黄酮醇存在于这两种提取物中。鉴于黄酮和黄酮醇是在花青素合成途径的中间步骤合成的,因此我们推测美洲黄莲中花青素合成途径的上游基因是有功能的,中断可能发生在花青素合成途径的下游。(2)通过简并PCR技术,从两物种中获得下游基因DFR1和ANS1。基于莲基因组的信息,我们构建本地blast。通过进行本地blast:和构建系统发育树,在莲中鉴定到参与花青素合成的4个UFGTs(UGT78A1-A4)和2个GSTs(GSTF11和GSTF12)基因。以红色花瓣的cDNA为模板进行扩增,检测到了4个UFGT的转录本和1个GST基因(GSTF11)的转录本。通过在两物种中比较这些结构基因的核苷酸序列及推测的氨基酸序列,发现这些结构基因具有较高的同源性,而且在已知的保守区和保守位点中几乎没有氨基酸差异。通过荧光实时定量PCR,发现DFR1、ANSI和UGT78A2在美洲黄莲的黄色花瓣中的表达水平高于莲红色花瓣中的表达水平,而剩下的3个UFGT基因在美洲黄莲花瓣中的表达水平低于莲花瓣中的表达水平。半定量PCR显示莲的花瓣中存在GSTs的转录本,而美洲黄莲的花瓣中不存在GSTs基因的转录本。GSTs基因转录本的缺失可能是美洲黄莲花瓣中不含花青素的关键原因。(3)通过本地blast,从莲基因组中总共鉴定到95个MYB基因,10个bHLH基因和17个WD40基因。通过与其他物种中已报道参与花青素合成的调节基因进行对比并构建系统发育树,我们发现莲中有9个MYB基因,2个bHLH基因和1个WD40基因(即TTG1)基因可能参与花青素合成途径的调控。以红色花瓣的cDNA为模板,通过PCR扩增,我们鉴定到1个MYB基因(即MYB5)和1个bHLH基因(即bHLH1)参与花瓣中花青素合成的调控。序列比较显示,在两物种中TTG1的氨基酸序列是一致的,而bHLH1只有1个氨基酸差异发生在bHLH结构域中。最显著的差异是MYB5基因,由于两个氨基酸的替代形成了一个提前终止密码子。对启动子区域潜在的顺式作用元件进行预测,结果显示美洲黄莲MYB5启动子有14种顺式作用元件,而莲MYB5中有13种。两物种的MYB5基因启动子中都包含很多涉及光响应的顺式作用元件。(4)居群水平上的研究揭示了MYB5基因有3种类型的等位基因。一种为有功能的等位基因,以纯合子的形式存在于莲的8个居群中；第二种为两个核苷酸替代(GAA/TAG)导致形成了一个提前终止密码子；第三种为一个核苷酸缺失导致形成了一个提前终止密码子。这两种无功能的等位基因只存在于美洲黄莲的居群中。其中第二种无义突变的等位基因存在于美洲黄莲的8个居群中,而第三种移码突变的等位基因存在于美洲黄莲的6个居群中。无义突变的等位基因有纯合子形式,而移码突变的等位基因总是和无义突变的等位基因一起,以杂合子形式存在,尚未检测到纯合子形式。(5)酵母双杂实验证明,莲中的NnMYB5不仅可以与莲中的NnbHLH1及NnTTG1互作,也可以与美洲黄莲中的N1bHLH1相互作用。而莲中NnTTGl也可以与两物种中的bHLH1相互作用。以上结果暗示,MYB5、bHLH1和TTG1可以形成MBW三元复合体。NnMYB5与NnbHLH1的相互作用强于与N1bHLH1的相互作用,表明bHLH1在两物种中的差异影响了其与MYB5的互作。(6)NnMYB5基因在拟南芥中的过表达导致花青素在未成熟的种子及花梗中积累,表明NnMYB5是花青素合成的转录激活子。拟南芥AtGsTF12基因在NnMYB5过表达植株中表达水平上调,表明AtGsTF12基因是NnMYB5的靶基因。由上述结果,推测出以下结论：莲与美洲黄莲的红、黄花色差异可能是由于两物种中的MYB5基因编码区的突变影响了其对结构基因的调节,进而影响了结构基因的表达水平与花青素的稳定积累所致。
[Abstract]:Lotus is a kind of ornamental, medicinal and edible aquatic plants. In one of the genus contains two species, namely the lotus (Nelumbo nucifera Gaertn.) and American Huang Lian (N. lutea Pers.). Although the two species discontinuously distributed in East Asia and North America and northern Australia, but there is no reproductive isolation, the hybrids. Besides, two species in the form of difference is not obvious, the most significant difference is the color. Lotus with red, pink, white and red and white four colors, no yellow, yellow. And America Huang Lian only molecular mechanism about the color of the lotus is very small, the only red, white color difference study, no differences the two flower species. What is the lotus and America Huang Lian what caused the close relationship in the color difference is so significant? In order to solve the mystery. In this study, the Heilongjiang wild lotus (red flower) and the American state of Florida Wild goldenseal (yellow flower) were studied. Firstly, spectrophotometer measurement red this study adopts the anthocyanin content in petals of yellow two. Then, through gene cloning, phylogenetic tree construction and local blast system, find the structural genes involved in anthocyanin synthesis and transport in the regulation and. The gene. By comparing nucleotide sequences of these genes in two species and the level of transcription, finding candidate genes may be involved in color difference, which is verified by yeast two hybrid and transgenic technology. The main results are as follows: (1) high content of anthocyanins in the wild lotus petals red, and yellow petals in goldenseal almost does not contain anthocyanin. This indicates that anthocyanin synthesis pathway in goldenseal petals could break. The wavelength scanning is displayed in the 350nm, two kinds of color of the petal extract has a high absorption Collect peak showed that flavones and flavonols exist in these two kinds of extracts. In view of flavonoids and flavonols are synthesized in the intermediate step of the anthocyanin synthesis pathway, so we speculate that the upstream genes of anthocyanin biosynthesis in goldenseal is a function of the interruption may occur in the anthocyanin synthesis pathway (2). By degenerate PCR, obtain the downstream gene DFR1 and ANS1. from two species of lotus genome based on information, we construct the local blast. through the local blast: and the phylogenetic tree, in the lotus 4 to participate in the identification of anthocyanin synthesis of UFGTs (UGT78A1-A4) and 2 GSTs (GSTF11 and GSTF12) gene in red. The petals of the cDNA was amplified, detected transcripts of 4 UFGT and 1 GST (GSTF11) gene transcripts. The amino acid sequence of nucleotide sequence comparison in two species of these structural genes and putative, The structure of genes with high homology in the conserved region, and known and conserved sites almost no amino acid difference. By real-time quantitative PCR, DFR1, the expression level of ANSI and UGT78A2 in goldenseal yellow petals in the above expression level in red lotus petals, and the expression level of 3 genes left UFGT in goldenseal petals in the lower expression level of lotus petals. The semi quantitative PCR showed the presence of GSTs transcripts of lotus petals, and goldenseal petals does not exist in the GSTs transcript of.GSTs gene transcript deletion may be a key reason of anthocyanin containing no goldenseal petals (3). Through the local blast, a total of 95 identified MYB gene from Lotus genome, 10 bHLH genes and 17 WD40 genes. With other species have been reported to be involved in the synthesis of anthocyanin regulatory gene for Ratio and phylogenetic tree, we found that there are 9 MYB genes in lotus, 2 bHLH genes and 1 WD40 genes (TTG1) gene may be involved in the biosynthesis of anthocyanins in red petals. CDNA as template, amplified by PCR, we identified 1 MYB genes (MYB5 and 1) a bHLH gene (bHLH1) is involved in the regulation of anthocyanin synthesis in petals. Sequence comparison showed that the amino acid sequence of TTG1 in the two species is the same, but bHLH1 has only 1 amino acid differences occurred in the bHLH domain. The most significant difference is due to the MYB5 gene, two amino acid substitution forms a a premature termination codon. The promoter region of potential cis acting elements were predicted. Results showed that goldenseal MYB5 promoter has 14 cis acting elements, and the lotus MYB5 13 species. Two species of MYB5 gene promoter contains many related CIS light response Effect of element. (4) in the study group on the level of the MYB5 gene revealed 3 types of alleles. A functional allele, YISHION zygote exists in the form of lotus of the 8 populations; second to two nucleotide substitutions (GAA/TAG) leads to the formation of a early termination codon third; a nucleotide deletion leads to the formation of a premature termination codon. These two kinds of function alleles exist only in goldenseal populations. Among the second kinds of nonsense mutation alleles exist in goldenseal 8 populations, and third a frameshift allele is present in goldenseal 6 populations. No allelic mutation is homozygous, and frameshift allele always and nonsense mutant alleles together, exist in the heterozygous form, has not detected the homozygous form (5). Yeast That two hybrid experiments, the lotus NnMYB5 can interact with NnbHLH1 and NnTTG1 in roots, but also with the goldenseal N1bHLH1 interaction. And the lotus NnTTGl can also interact with the two species of bHLH1. These results suggested that MYB5, bHLH1 and TTG1 can form MBW complex with.NnMYB5 three yuan the interaction of NnbHLH1 in strong interaction with N1bHLH1 and bHLH1 in two species showed differences in the effects of interaction with MYB5. (6) NnMYB5 gene over expression in Arabidopsis thaliana resulted in anthocyanin accumulation in immature seeds and pedicel, indicate that NnMYB5 is a transcriptional activator of anthocyanin biosynthesis in Arabidopsis AtGsTF12 gene. Over expression was up-regulated in plants in NnMYB5, suggesting that the AtGsTF12 gene is the target gene of NnMYB5. From the results that the following conclusions: lotus and goldenseal red, yellow color difference may be due to two species The mutation of the MYB5 gene coding region affects the regulation of the structural genes, and then affects the expression level of the structural genes and the stable accumulation of anthocyanins.

【学位授予单位】：中国科学院研究生院(武汉植物园)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S682.32;Q943.2

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